Epigenetic dysregulation from chromosomal transit in micronuclei

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers1–4, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei5,6 and subsequent rupture of the micronuclear envelope7 profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed. Using orthogonal approaches, we demonstrate that micronuclei exhibit extensive differences in chromatin accessibility, with a strong positional bias between promoters and distal or intergenic regions, in line with observed redistributions of histone PTMs. Inducing CIN causes widespread epigenetic dysregulation, and chromosomes that transit in micronuclei experience heritable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, as well as altering genomic copy number, CIN promotes epigenetic reprogramming and heterogeneity in cancer.

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Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy The ATAC-Seq, CUT and RUN and WGS generated in this study is publicly available (GEO accession code accession code GSE186589, SRA accession code PRJNA882761, ). Mass spectrometry raw files are deposited in the repository Chorus (https://chorusproject.org/) under the project number 1790. WGS files from EGA (dataset ID EGAD0000100416) was used in DLD-1 CEN-SELECT samples analysis. The TCGA dataset used was Breast Invasive Carcinoma (TCGA, PanCancer Atlas, https://www.cbioportal.org/study/summary?id=brca_tcga_pan_can_atlas_2018).

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Life sciences study design
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Sample size
No statistical methods were used to pre-determine sample size. Sample sizes were determined based on prior experience and were chosen as more than or equal to three biological or technical replicates. For micronuclei ATAC-Seq and CUT&RUN, biological duplicates were performed.
Data exclusions In FLIM experiment where MATLAB function "isooutlier" was used to clear outliers. Outliers (defined as elements more than 1.5 interquartile ranges above the upper quartile or below the lower quartile) were excluded to remove inconsistent lifetime values caused by poor signal-tonoise ratio. There was no exclusion in all other data.

Replication
Experimental and biological replicates are mentioned in figure legends and methods section.
Randomization Not applicable to this study as no intervention was performed, therefore, it is not possible to randomize.

Blinding
Investigator was not blinded as difference was clear from staining as well as morphology for micronuclei counting and chromosomal missegregation scoring. Therefore blinding was not possible in the experiments.
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Validation
The primary antibodies used were purchased from reputable sources validated for the species and application (immunoblotting and/ or immunofluorescence) by their respective manufacturers in their website's validation statements. The validations were done using recombinant protein and/or cell lines known to express the target protein as a positive control. Moreover antibodies for H3K27me3, H3K14ac, H3K27ac was futher validated using pharmacological inhibition in this manuscript as show in figures 1c and extended data figure 2b.

Authentication
All cell lines used in this manuscript were authenticated by ATCC which used morphology, karyotyping and PCR-based techniques.
Mycoplasma contamination All cell lines tested negative for mycoplasma according to the test kit from R&D Systems™ (MycoProbe Mycoplasma Detection Kit).

Human research participants
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Population characteristics
The study cohort (MSK SPECTRUM) includes 42 women with newly diagnosed, treatment-naive high-grade serous ovarian cancer (HGSOC). Patients between the ages of 39 and 81 at diagnosis (median age: 61 years). 6 out of 42 cases had BRCA1 mutations (14%) and 1 out of 42 cases had a BRCA2 mutation (2%).

Recruitment
All enrolled patients were consented to an institutional biospecimen banking protocol and a protocol to perform targetted panel sequencing (MSK-IMPACT). All analyses were performed per a biospecimen research protocol. All protocols were approved by the Institutional Review Board (IRB) of Memorial Sloan Kettering Cancer Center (MSKCC). Patients were consented following the IRB-approved standard operating procedures for informed consent. Written informed consent was obtained from all patients before conducting any study-related procedures. This study was conducted in accordance with the Declaration of Helsinki and the Good Clinical Practice guidelines (GCP).

March 2021
Ethics oversight Institutional Review Board (IRB) at Memorial Sloan Kettering Cancer Center (MSKCC).
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Flow Cytometry
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Methodology Sample preparation
Mentioned in the method section under "micronuclei purification". Briefly, cells were expanded in 245 × 245 × 25 mm dishes and treated with reversine as previously specified. Cells were then harvested and washed DMEM. Washed cells were resuspended in pre-warmed (37 °C) DMEM supplemented with 10 μg/ml cytochalasin B (Sigma-Aldrich) at a concentration of around 5x106 cells/mL DMEM and incubated at 37°C for at least 30 minutes. Subsequently, cells were centrifuged at 300g for 5 minutes and resuspended in cold lysis buffer (10 mM Tris-HCl, 2 mM Mg-acetate, 3 mM CaCl2, 0.32 M sucrose, 0.1 mM EDTA, 0.1 % (v/v) NP-40, pH 8.5) freshly complemented (with 1 mM dithiothreitol, 0.15 mM spermine, 0.75 mM spermidine, 10 μg/ml cytochalasin B and protease inhibitors) at a concentration of 107 cells/ml lysis buffer. Resuspended cells were then dounce-homogenized 10 times using a loose-fitting pestle. Then, lysates were mixed with an equal volume of cold 1.8 M sucrose buffer (10 mM Tris-HCl, 1.8 M sucrose, 5 mM Mg-acetate, 0.1 mM EDTA, pH 8.0) freshly complemented (with 1 mM dithiothreitol, 0.3 % BSA, 0.15 mM spermine, 0.75 mM spermidine) before use. In a 50 mL conical tube, 10 mL of the mixture was then layered on top of a two-layer sucrose gradient (20 mL of 1.8 M sucrose buffer on top of 15 mL 1.6 M sucrose buffer). This mixture was then centrifuged in a swing bucket centrifuge at 950g for 20 min at 4°C. The first resulting 2 mL top fraction is discarded; next 5-6 mL mostly contain micronuclei is collected, the next 3 mL mostly containing primary nuclei is also collected in a separate container. Collected fractions were diluted 1:5 with FACS buffer (ice cold PBS freshly supplemented with 0.3 % BSA, 0.1 % NP-40 and protease inhibitors). Diluted MN were then centrifuged at 950g in JS-5.2 swing bucket centrifuge for 20 min at 4 °C. The resulting supernatant was removed by aspiration and either micronuclei or primary nuclei was resuspended in 2-4 mL of FACS buffer supplemented with 2 μg/ml DAPI (however, no DAPI was used for micronuclei purification for ATAC-seq experiments). Resuspended samples were filtered through a 40 μm ministrainer (PluriSelect) into FACS tubes.

Instrument
FACSAria III Cell Sorter Software FlowJo Cell population abundance N/A (Nuclei was sorted instead of cells)

Gating strategy
Mentioned in the method section under "micronuclei purification". Micronuclei were sorted by FACSAria (BD Biosciences) into FACS buffer at the MSKCC Flow Cytometry Core Facility. Default FSC and DAPI thresholds were lowered, and a log scale was used to visualize MN population. Population in the area that has lower FSC and DAPI signals is gated for micronuclei while higher FSC and DAPI signals is gated for primary nuclei. From micronuclei gate, the population in the area that has mCherry positive, low GFP signal is gated for intact micronuclei, while the higher GFP signal is gated for ruptured micronuclei. The figure exemplifying the gating strategy is provided in extended data figure 6e.
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